Adeno-associated virus serotype I nucleic acid sequences, vectors and host cells containing same

ABSTRACT

The nucleic acid sequences of adeno-associated virus (AAV) serotype 1 are provided, as are vectors and host cells containing these sequences and functional fragments thereof. Also provided are methods of delivering genes via AAV-1 derived vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 14/136,331,filed Dec. 20, 2013, which is a continuation of U.S. patent applicationSer. No. 13/048,936, filed Mar. 16, 2011, now U.S. Pat. No. 8,637,255,which is a continuation of U.S. patent application Ser. No. 12/617,967,filed Nov. 13, 2009, now abandoned, which is a continuation of U.S.patent application Ser. No. 11/893,697, filed Aug. 17, 2007, nowabandoned, which is a continuation of U.S. patent application Ser. No.11/708,785, filed Feb. 20, 2007, now abandoned, which is a continuationof U.S. patent application Ser. No. 10/696,900, filed Oct. 30, 2003, nowU.S. Pat. No. 7,186,552, which is a continuation of U.S. patentapplication Ser. No. 09/807,802, filed Nov. 29, 2001, now U.S. Pat. No.6,759,237, which is a national stage under 35 USC 371 of PCT/US99/25694,filed Nov. 2, 1999, which claims the benefit under 35 USC 119(e) of thepriority of U.S. Patent Application No. 60/107,114, filed Nov. 5, 1998.

U.S. patent application Ser. No. 11/893,697, filed Aug. 17, 2007, nowabandoned, is also a continuation of U.S. patent application Ser. No.11/430,226, filed May 8, 2006, now abandoned, which is a divisional ofU.S. patent application Ser. No. 10/696,282, filed Oct. 29, 2003, nowU.S. Pat. No. 7,105,345, which is a divisional of U.S. patentapplication Ser. No. 09/807,802, filed Nov. 29, 2001, now U.S. Pat. No.6,759,237, which is a national stage of PCT/US99/25694, filed Nov. 2,1999, which claims the benefit of the priority of U.S. PatentApplication No. 60/107,114, filed Nov. 5, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by the National Institutes of Health, grant no.P30 DK47757-06 and PO1 HD32649-04. The US government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

This invention relates generally to viral vector, and more particularly,to recombinant viral vectors useful for gene delivery.

Adeno-associated viruses are small, single-stranded DNA viruses whichrequire helper virus to facilitate efficient replication [K. I. Berns,Parvoviridae: the viruses and their replication, p. 1007-1041, in F. N.Fields et al., Fundamental virology, 3rd ed., vol. 2, (Lippencott-RavenPublishers, Philadelphia, Pa.) (1995)]. The 4.7 kb genome of AAV ischaracterized by two inverted terminal repeats (ITR) and two openreading frames which encode the Rep proteins and Cap proteins,respectively. The Rep reading frame encodes four proteins of molecularweight 78 kD, 68 kD, 52 kD and 40 kD. These proteins function mainly inregulating AAV replication and integration of the AAV into a host cell'schromosomes. The Cap reading frame encodes three structural proteins inmolecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3) [Berns, citedabove]. More than 80% of total proteins in AAV virion comprise VP3. Thetwo ITRs are the only cis elements essential for AAV replication,packaging and integration. There are two conformations of AAV ITRscalled “flip” and “flop”. These differences in conformation originatedfrom the replication model of adeno-associated virus which use the ITRto initiate and reinitiate the replication [R. O. Snyder et al., J.Virol., 67:6096-6104 (1993); K. I. Berns, Microbiological Reviews,54:316-329 (1990)].

AAVs have been found in many animal species, including primates, canine,fowl and human [F. A. Murphy et al., “The Classification andNomenclature of Viruses: Sixth Report of the International Committee onTaxonomy of Viruses”, Archives of Virology, (Springer-Verlag, Vienna)(1995)]. In addition to five known primate AAVs (AAV-1 to AAV-5), AAV-6,another serotype closely related to AAV-2 and AAV-1 has also beenisolated [E. A. Rutledge et al., J. Virol., 72:309-319 (1998)]. Amongall known AAV serotypes, AAV-2 is perhaps the most well-characterizedserotype, because its infectious clone was the first made [R. J.Samulski et al., Proc. Natl. Acad. Sci. USA, 79:2077-2081 (1982)].Subsequently, the full sequences for AAV-3A, AAV-3B, AAV-4 and AAV-6have also been determined [Rutledge, cited above; J. A. Chiorini et al.,J. Virol., 71:6823-6833 (1997); S. Muramatsu et al., Virol., 221:208-217(1996)]. Generally, all AAVs share more than 80% homology in nucleotidesequence.

A number of unique properties make AAV a promising vector for human genetherapy [Muzyczka, Current Topics in Microbiology and Immunology,158:97-129 (1992)]. Unlike other viral vectors, AAVs have not been shownto be associated with any known human disease and are generally notconsidered pathogenic. Wild type AAV is capable of integrating into hostchromosomes in a site specific manner [R. M. Kotin et al., Proc. Natl.Acad. Sci. USA, 87:2211-2215 (1990); R. J. Samulski, EMBO J.,10(12):3941-3950 (1991)]. Recombinant AAV vectors can integrate intotissue cultured cells in chromosome 19 if the rep proteins are suppliedin trans [C. Balague et al., J. Virol., 71:3299-3306 (1997); R. T.Surosky et al., J. Virol., 71:7951-7959 (1997)]. The integrated genomesof AAV have been shown to allow long term gene expression in a number oftissues, including, muscle, liver, and brain [K. J. Fisher, Nature Med.,3(3):306-312 (1997); R. 0. Snyder et al., Nature Genetics, 16:270-276(1997); X. Xiao et al., Experimental Neurology, 144:113-124 (1997);Xiao, J. Virol., 70(11):8098-8108 (1996)].

AAV-2 has been shown to be present in about 80-90% of the humanpopulation. Earlier studies showed that neutralizing antibodies forAAV-2 are prevalent [W. P. Parks et al., J. Virol., 2:716-722 (1970)].The presence of such antibodies may significantly decrease theusefulness of AAV vectors based on AAV-2 despite its other merits. Whatare needed in the art are vectors characterized by the advantages ofAAV-2, including those described above, without the disadvantages,including the presence of neutralizing antibodies.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated AAV-1 nucleic acidmolecule which is selected from among SEQ ID NO: 1, the strandcomplementary to SEQ ID NO: 1, and cDNA and RNA sequences complementaryto SEQ ID NO: 1 and its complementary strand.

In another aspect, the present invention provides AAV ITR sequences,which include the 5′ ITR sequences, nt 1 to 143 of SEQ ID NO: 1; the 3′ITR sequences, nt 4576 to 4718 of SEQ ID NO: 1, and fragments thereof.

In yet another aspect, the present invention provides a recombinantvector comprising an AAV-1 ITR and a selected transgene. Preferably, thevector comprises both the 5′ and 3′ AAV-1 ITRs between which theselected transgene is located.

In still another aspect, the invention provides a recombinant vectorcomprising an AAV-1 P5 promoter having the sequence of nt 236 to 299 ofSEQ ID NO: 1 or a functional fragment thereof.

In a further aspect, the present invention provides a nucleic acidmolecule encoding an AAV-1 rep coding region and an AAV-1 cap codingregion.

In still another aspect, the present invention provides a host celltransduced with a recombinant viral vector of the invention. Theinvention further provides a host cell stably transduced with an AAV-1P5 promoter of the invention.

In still a further aspect, the present invention provides apharmaceutical composition comprising a carrier and a vector of theinvention.

In yet another aspect, the present invention provides a method for AAVmediated delivery of a transgene to a host involving the step ofdelivering to a selected host a recombinant viral vector comprising aselected transgene under the control of sequences which directexpression thereof and an adeno-associated virus 1 (AAV-1) virion.

In another aspect, the invention provides a method for in vitroproduction of a selected gene product using a vector of the invention.

Other aspects and advantages of the invention will be readily apparentto one of skill in the art from the detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F illustrate the alignment of nucleotides of AAV-1 [SEQ ID NO:1], AAV-2 [SEQ ID NO: 18] and AAV-6 [SEQ ID NO: 19]. The alignment wasdone with MacVector 6.0. The full sequences of AAV-1 are shown in thetop line. Nucleotides in AAV-2 and AAV-6 identical to AAV-1 aresymbolized by “.” and gaps by “-”. Some of the conserved features amongAAVs are marked in this figure. Note the 3′ ITRs of AAV-1 and AAV-6 areshown in different orientations.

FIG. 2 illustrates the predicted secondary structure of AAV-1 ITR (nt1-146 of SEQ ID NO:1). The nucleotides in AAV-2 (nt 1-144 of SEQ IDNO:18) and AAV-6 (nt 1-136 of SEQ ID NO:19) are shown in italic and boldrespectively.

FIG. 3A illustrates a hypothesis of how AAV-6 arose from the homologousrecombination between AAV-1 and AAV-2. The major elements of AAV-1 areindicated in the graph. A region that is shared between AAV-1, AAV-2 andAAV-6 is shown in box with waved lines.

FIG. 3B is a detailed illustration of a 71 bp homologous region amongAAV-1 (438-531 of SEQ ID NO:1), AAV-2 (424-513 of SEQ ID NO:18) andAAV-6 (423-512 of SEQ ID NO:19). Nucleotides that differ among theseserotypes are indicated by arrows.

FIG. 4A is a bar chart illustrating expression levels of human alpha 1anti-trypsin (α1AT) in serum following delivery of hAAT via recombinantAAV-1 and recombinant AAV-2 viruses.

FIG. 4B is a bar chart illustrating expression levels of erythropoietin(epo) in serum following delivery of the epo gene via recombinant AAV-1and recombinant AAV-2 viruses.

FIG. 5A is a bar chart illustrating expression levels of α1AT in liverfollowing delivery of α1AT as described in Example 7.

FIG. 5B is a bar chart demonstrating expression levels of epo in liverfollowing delivery of epo as described in Example 7.

FIG. 5C is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-1 following delivery of α1AT or epo to liver asdescribed in Example 7.

FIG. 5D is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-2 following delivery of α1AT or epo to liver asdescribed in Example 7.

FIG. 6A is a bar chart illustrating expression levels of α1AT in musclefollowing delivery of α1AT as described in Example 7.

FIG. 6B is a bar chart demonstrating expression levels of epo in musclefollowing delivery of epo as described in Example 7.

FIG. 6C is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-1 following delivery of α1AT or epo to muscle asdescribed in Example 7.

FIG. 6D is a bar chart demonstrating neutralizing antibodies (NAB)directed to AAV-2 following delivery of α1AT or epo to muscle asdescribed in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel nucleic acid sequences for anadeno-associated virus of serotype 1 (AAV-1). Also provided arefragments of these AAV-1 sequences. Among particularly desirable AAV-1fragments are the inverted terminal repeat sequences (ITRs), rep andcap. Each of these fragments may be readily utilized, e.g., as acassette, in a variety of vector systems and host cells. Such fragmentsmay be used alone, in combination with other AAV-1 sequences orfragments, or in combination with elements from other AAV or non-AAVviral sequences. In one particularly desirable embodiment, a cassettemay contain the AAV-1 ITRs of the invention flanking a selectedtransgene. In another desirable embodiment, a cassette may contain theAAV-1 rep and/or cap proteins, e.g., for use in producing recombinant(rAAV) virus.

Thus, the AAV-1 sequences and fragments thereof are useful in productionof rAAV, and are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. The invention further provides nucleic acidmolecules, gene delivery vectors, and host cells which contain the AAV-1sequences of the invention. Also provided a novel methods of genedelivery using AAV vectors.

As described herein, the vectors of the invention containing the AAV-1capsid proteins of the invention are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other AAV serotype based vectors, as well as otherviral vectors. The rAAV vectors of the invention are particularlyadvantageous in rAAV readministration and repeat gene therapy.

These and other embodiments and advantages of the invention aredescribed in more detail below. As used throughout this specificationand the claims, the term “comprising” is inclusive of other components,elements, integers, steps and the like.

I. AAV1 Nucleic Acid and Protein Sequences

The AAV-1 nucleic acid sequences of the invention include the DNAsequences of SEQ ID NO: 1 (FIGS. 1A-1F), which consists of 4718nucleotides. The AAV-1 nucleic acid sequences of the invention furtherencompass the strand which is complementary to SEQ ID NO: 1, as well asthe RNA and cDNA sequences corresponding to SEQ ID NO: 1 and itscomplementary strand. Also included in the nucleic acid sequences of theinvention are natural variants and engineered modifications of SEQ IDNO: 1 and its complementary strand. Such modifications include, forexample, labels which are known in the art, methylation, andsubstitution of one or more of the naturally occurring nucleotides withan analog.

Further included in this invention are nucleic acid sequences which aregreater than 85%, preferably at least about 90%, more preferably atleast about 95%, and most preferably at least about 98-99% identical orhomologous to SEQ ID NO:1.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length sequence, or afragment at least about nine nucleotides, usually at least about 20-24nucleotides, at least about 28-32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFasta, a program in GCG Version 6.1. Fasta provides alignments andpercent sequence identity of the regions of the best overlap between thequery and search sequences (Pearson, 1990, herein incorporated byreference). For instance, percent sequence identity between nucleic acidsequences can be determined using Fasta with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) as providedin GCG Version 6.1, herein incorporated by reference.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95-99% of the sequence.

Also included within the invention are fragments of SEQ ID NO: 1, itscomplementary strand, cDNA and RNA complementary thereto. Suitablefragments are at least 15 nucleotides in length, and encompassfunctional fragments which are of biological interest. Certain of thesefragments may be identified by reference to FIGS. 1A-1F. Examples ofparticularly desirable functional fragments include the AAV-1 invertedterminal repeat (ITR) sequences of the invention. In contrast to the 145nt ITRs of AAV-2, AAV-3, and AAV-4, the AAV-1 ITRs have been found toconsist of only 143 nucleotides, yet advantageously are characterized bythe T-shaped hairpin structure which is believed to be responsible forthe ability of the AAV-2 ITRs to direct site-specific integration. Inaddition, AAV-1 is unique among other AAV serotypes, in that the 5′ and3′ ITRs are identical. The full-length 5′ ITR sequences of AAV-1 areprovided at nucleotides 1-143 of SEQ ID NO: 1 (FIG. 1A) and thefull-length 3′ ITR sequences of AAV-1 are provided at nt 4576-4718 ofSEQ ID NO: 1 (FIG. 1F). One of skill in the art can readily utilize lessthan the full-length 5′ and/or 3′ ITR sequences for various purposes andmay construct modified ITRs using conventional techniques, e.g., asdescribed for AAV-2 ITRs in Samulski et al, Cell, 33:135-143 (1983).

Another desirable functional fragment of the AAV-1 genome is the P5promoter of AAV-1 which has sequences unique among AAV P5 promoters,while maintaining critical regulatory elements and functions. Thispromoter is located within nt 236-299 of SEQ ID NO: 1 (FIG. 1A). Otherexamples of functional fragments of interest include the sequences atthe junction of the rep/cap, e.g., the sequences spanning nt 2306-2223,as well as larger fragments which encompass this junction which maycomprise 50 nucleotides on either side of this junction. Still otherexamples of functional fragments include the sequences encoding the repproteins. Rep 78 is located in the region of nt 334-2306 of SEQ ID NO:1; Rep 68 is located in the region of nt 334-2272, and contains anintron spanning nt 1924-2220 of SEQ ID NO: 1. Rep 52 is located in theregion of nt 1007-2304 of SEQ ID NO: 1; rep 40 is located in the regionof nt 1007-2272, and contains an intron spanning nt 1924-2246 of SEQ IDNO: 1. Also of interest are the sequences encoding the capsid proteins,VP 1 [nt 2223-4431 of SEQ ID NO: 1], VP2 [nt 2634-4432 of SEQ ID NO: 1]and VP3 [nt 2829-4432 of SEQ ID NO: 1]. Other fragments of interest mayinclude the AAV-1 P19 sequences, AAV-1 P40 sequences, the rep bindingsite, and the terminal resolute site (TRS).

The invention further provides the proteins and fragments thereof whichare encoded by the AAV-1 nucleic acids of the invention. Particularlydesirable proteins include the rep and cap proteins, which are encodedby the nucleotide sequences identified above. These proteins include rep78 [SEQ ID NO:5], rep 68 [SEQ ID NO:7], rep 52 [SEQ ID NO:9], rep 40[SEQ ID NO: 11], vp1 [SEQ ID NO: 13], vp2 [SEQ ID NO: 15], and vp3 [SEQID NO: 17] and functional fragments thereof while the sequences of therep and cap proteins have been found to be closely related to those ofAAV-6, there are differences in the amino acid sequences (see Table 1below), as well as differences in the recognition of these proteins bythe immune system. However, one of skill in the art may readily selectother suitable proteins or protein fragments of biological interest.Suitably, such fragments are at least 8 amino acids in length. However,fragments of other desired lengths may be readily utilized. Suchfragments may be produced recombinantly or by other suitable means,e.g., chemical synthesis.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Such production methods are withinthe knowledge of those of skill in the art and are not a limitation ofthe present invention.

II. Viral Vectors

In another aspect, the present invention provides vectors which utilizethe AAV-1 sequences of the invention, including fragments thereof, fordelivery of a heterologous gene or other nucleic acid sequences to atarget cell. Suitably, these heterologous sequences (i.e., a transgene)encode a protein or gene product which is capable of being expressed inthe target cell. Such a transgene may be constructed in the form of a“minigene”. Such a “minigene” includes selected heterologous genesequences and the other regulatory elements necessary to transcribe thegene and express the gene product in a host cell. Thus, the genesequences are operatively linked to regulatory components in a mannerwhich permit their transcription. Such components include conventionalregulatory elements necessary to drive expression of the transgene in acell containing the viral vector. The minigene may also contain aselected promoter which is linked to the transgene and located, withother regulatory elements, within the selected viral sequences of therecombinant vector.

Selection of the promoter is a routine matter and is not a limitation ofthis invention. Useful promoters may be constitutive promoters orregulated (inducible) promoters, which will enable control of the timingand amount of the transgene to be expressed. For example, desirablepromoters include the cytomegalovirus (CMV) immediate earlypromoter/enhancer [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the Rous sarcoma virus LTR promoter/enhancer, and the chickencytoplasmic β-actin promoter [T. A. Kost et al, Nucl. Acids Res.,11(23):8287 (1983)]. Still other desirable promoters are the albuminpromoter and an AAV P5 promoter. Optionally, the selected promoter isused in conjunction with a heterologous enhancer, e.g., the β-actinpromoter may be used in conjunction with the CMV enhancer. Yet othersuitable or desirable promoters and enhancers may be selected by one ofskill in the art.

The minigene may also desirably contain nucleic acid sequencesheterologous to the viral vector sequences including sequences providingsignals required for efficient polyadenylation of the transcript (poly-Aor pA) and introns with functional splice donor and acceptor sites. Acommon poly-A sequence which is employed in the exemplary vectors ofthis invention is that derived from the papovavirus SV-40. The poly-Asequence generally is inserted in the minigene downstream of thetransgene sequences and upstream of the viral vector sequences. A commonintron sequence is also derived from SV-40, and is referred to as theSV40 T intron sequence. A minigene of the present invention may alsocontain such an intron, desirably located between the promoter/enhancersequence and the transgene. Selection of these and other common vectorelements are conventional [see, e.g., Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d edit., Cold Spring Harbor Laboratory,New York (1989) and references cited therein] and many such sequencesare available from commercial and industrial sources as well as fromGenbank.

The selection of the transgene is not a limitation of the presentinvention. Suitable transgenes may be readily selected from amongdesirable reporter genes, therapeutic genes, and optionally, genesencoding immunogenic polypeptides. Examples of suitable reporter genesinclude (3-galactosidase (β-gal), an alkaline phosphatase gene, andgreen fluorescent protein (GFP). Examples of therapeutic genes include,cytokines, growth factors, hormones, and differentiation factors, amongothers. The transgene may be readily selected by one of skill in theart. See, e.g., WO 98/09657, which identifies other suitable transgenes.

Suitably, the vectors of the invention contain, at a minimum, cassetteswhich consist of fragments of the AAV-1 sequences and proteins. In oneembodiment, a vector of the invention comprises a selected transgene,which is flanked by a 5′ ITR and a 3′ ITR, at least one of which is anAAV-1 ITR of the invention. Suitably, vectors of the invention maycontain a AAV-1 P5 promoter of the invention. In yet another embodiment,a plasmid or vector of the invention contains AAV-1 rep sequences. Instill another embodiment, a plasmid or vector of the invention containsat least one of the AAV-1 cap proteins of the invention. Most suitably,these AAV-1-derived vectors are assembled into viral vectors, asdescribed herein.

A. AAV Viral Vectors

In one aspect, the present invention provides a recombinant AAV-1 viralvector produced using the AAV-1 capsid proteins of the invention. Thepackaged rAAV-1 virions of the invention may contain, in addition to aselected minigene, other AAV-1 sequences, or may contain sequences fromother AAV serotypes.

Methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, J. Viral., 70:520-532 (1993) and U.S. Pat. No.5,478,745. In one suitable method, a selected host cell is provided withthe AAV sequence encoding a rep protein, the gene encoding the AAV capprotein and with the sequences for packaging and subsequent delivery.Desirably, the method utilizes the sequences encoding the AAV-1 repand/or cap proteins of the invention.

In one embodiment, the rep/cap genes and the sequences for delivery aresupplied by co-transfection of vectors carrying these genes andsequences. In one currently preferred embodiment, a cis (vector)plasmid, a trans plasmid containing the rep and cap genes, and a plasmidcontaining the adenovirus helper genes are co-transfected into asuitable cell line, e.g., 293. Alternatively, one or more of thesefunctions may be provided in trans via separate vectors, or may be foundin a suitably engineered packaging cell line.

An exemplary cis plasmid will contain, in 5′ to 3′ order, AAV 5′ ITR,the selected transgene, and AAV 3′ ITR. In one desirable embodiment, atleast one of the AAV ITRs is a 143 nt AAV-1 ITR. However, other AAVserotype ITRs may be readily selected. Suitably, the full-length ITRsare utilized. However, one of skill in the art can readily preparemodified AAV ITRs using conventional techniques. Similarly, methods forconstruction of such plasmids is well known to those of skill in theart.

A trans plasmid for use in the production of the rAAV-1 virion particlemay be prepared according to known techniques. In one desiredembodiment, this plasmid contains the rep and cap proteins of AAV-1, orfunctional fragments thereof. Alternatively, the rep sequences may befrom another selected AAV serotype.

The cis and trans plasmid may then be co-transfected with a wild-typehelper virus (e.g., Ad2, Ad5, or a herpesvirus), or more desirably, areplication defective adenovirus, into a selected host cell.Alternatively, the cis and trans plasmid may be co-transfected into aselected host cell together with a transfected plasmid which providesthe necessary helper functions. Selection of a suitable host cell iswell within the skill of those in the art and include such mammaliancells as 293 cells, HeLa cells, among others.

Alternatively, the cis plasmid and, optionally the trans plasmid, may betransfected into a packaging cell line which provides the remaininghelper functions necessary for production of a rAAV containing thedesired AAV-1 sequences of the invention. An example of a suitablepackaging cell line, where an AAV-2 capsid is desired, is B-50, whichstably expresses AAV-2 rep and cap genes under the control of ahomologous P5 promoter. This cell line is characterized by integrationinto the cellular chromosome of multiple copies (at least 5 copies) ofP5-rep-cap gene cassettes in a concatomer form. This B-50 cell line wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Sep. 18, 1997 under AccessionNo. CRL-12401 pursuant to the provisions of the Budapest Treaty.However, the present invention is not limited as to the selection of thepackaging cell line.

Exemplary transducing vectors based on AAV-1 capsid proteins have beentested both in vivo and in vitro, as described in more detail in Example4. In these studies, it was demonstrated that recombinant AAV vectorwith an AAV-1 virion can transduce both mouse liver and muscle. These,and other AAV-1 based gene therapy vectors which may be generated by oneof skill in the art are beneficial for gene delivery to selected hostcells and gene therapy patients since the neutralization antibodies ofAAV-1 present in much of the human population exhibit different patternsfrom other AAV serotypes and therefore do not neutralize the AAV-1virions. One of skill in the art may readily prepare other rAAV viralvectors containing the AAV-1 capsid proteins provided herein using avariety of techniques known to those of skill in the art. One maysimilarly prepare still other rAAV viral vectors containing AAV-1sequence and AAV capsids of another serotype.

B. Other Viral Vectors

One of skill in the art will readily understand that the AAV-1 sequencesof the invention can be readily adapted for use in these and other viralvector systems for in vitro, ex vivo or in vivo gene delivery.Particularly well suited for use in such viral vector systems are theAAV-1 ITR sequences, the AAV-1 rep, the AAV-1 cap, and the AAV-1 P5promoter sequences.

For example, in one desirable embodiment, the AAV-1 ITR sequences of theinvention may be used in an expression cassette which includes AAV-1 5′ITR, a non-AAV DNA sequences of interest (e.g., a minigene), and 3′ ITRand which lacks functional rep/cap. Such a cassette containing an AAV-1ITR may be located on a plasmid for subsequent transfection into adesired host cell, such as the cis plasmid described above. Thisexpression cassette may further be provided with an AAV capsid of aselected serotype to permit infection of a cell or stably transfectedinto a desired host cell for packaging of rAAV virions. Such anexpression cassette may be readily adapted for use in other viralsystems, including adenovirus systems and lentivirus systems. Methods ofproducing Ad/AAV vectors are well known to those of skill in the art.One desirable method is described in PCT/US95/14018. However, thepresent invention is not limited to any particular method.

Another aspect of the present invention is the novel AAV-1 P5 promotersequences which are located in the region spanning nt 236-299 of SEQ IDNO: 1. This promoter is useful in a variety of viral vectors for drivingexpression of a desired transgene.

Similarly, one of skill in the art can readily select other fragments ofthe AAV-1 genome of the invention for use in a variety of vectorsystems. Such vectors systems may include, e.g., lentiviruses,retroviruses, poxviruses, vaccinia viruses, and adenoviral systems,among others. Selection of these vector systems is not a limitation ofthe present invention.

C. Host Cells and Packaging Cell Lines

In yet another aspect, the present invention provides host cells whichmay be transiently transfected with AAV-1 nucleic acid sequences of theinvention to permit expression of a desired transgene or production of arAAV particle. For example, a selected host cell may be transfected withthe AAV-1 P5 promoter sequences and/or the AAV-1 5′ ITR sequences usingconventional techniques. Providing AAV helper functions to thetransfected cell lines of the invention results in packaging of the rAAVas infectious rAAV particles. Such cell lines may be produced inaccordance with known techniques [see, e.g, U.S. Pat. No. 5,658,785],making use of the AAV-1 sequences of the invention.

Alternatively, host cells of the invention may be stably transfectedwith a rAAV expression cassette of the invention, and with copies ofAAV-1 rep and cap genes. Suitable parental cell lines include mammaliancell lines and it may be desirable to select host cells from amongnon-simian mammalian cells. Examples of suitable parental cell linesinclude, without limitation, HeLa [ATCC CCL 2], A549 [ATCC Accession No.CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI38 [CCL75] cells. These cell lines are all available from the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209USA. Other suitable parent cell lines may be obtained from other sourcesand may be used to construct stable cell lines containing the P5 and/orAAV rep and cap sequences of the invention.

Recombinant vectors generated as described above are useful for deliveryof the DNA of interest to cells.

III. Methods of Delivering Genes Via AAV-1 Derived Vectors

In another aspect, the present invention provides a method for deliveryof a transgene to a host which involves transfecting or infecting aselected host cell with a recombinant viral vector generated with theAAV-1 sequences (or functional fragments thereof) of the invention.Methods for delivery are well known to those of skill in the art and arenot a limitation of the present invention.

In one desirable embodiment, the invention provides a method forAAV-mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequenceswhich direct expression thereof and AAV-1 capsid proteins.

Optionally, a sample from the host may be first assayed for the presenceof antibodies to a selected AAV serotype. A variety of assay formats fordetecting neutralizing antibodies are well known to those of skill inthe art. The selection of such an assay is not a limitation of thepresent invention. See, e.g., Fisher et al, Nature Med., 3(3):306-312(March 1997) and W. C. Manning et al, Human Gene Therapy, 9:477-485(Mar. 1, 1998). The results of this assay may be used to determine whichAAV vector containing capsid proteins of a particular serotype arepreferred for delivery, e.g., by the absence of neutralizing antibodiesspecific for that capsid serotype.

In one aspect of this method, the delivery of vector with AAV-1 capsidproteins may precede or follow delivery of a gene via a vector with adifferent serotype AAV capsid protein. Thus, gene delivery via rAAVvectors may be used for repeat gene delivery to a selected host cell.Desirably, subsequently administered rAAV vectors carry the sametransgene as the first rAAV vector, but the subsequently administeredvectors contain capsid proteins of serotypes which differ from the firstvector. For example, if a first vector has AAV-2 capsid proteins,subsequently administered vectors may have capsid proteins selected fromamong the other serotypes, including AAV-1, AAV-3A, AAV-3B, AAV-4 andAAV-6.

Thus, a rAAV-1-derived recombinant viral vector of the inventionprovides an efficient gene transfer vehicle which can deliver a selectedtransgene to a selected host cell in vivo or ex vivo even where theorganism has neutralizing antibodies to one or more AAV serotypes. Thesecompositions are particularly well suited to gene delivery fortherapeutic purposes. However, the compositions of the invention mayalso be useful in immunization. Further, the compositions of theinvention may also be used for production of a desired gene product invitro.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. An AAV viral vector bearing the selectedtransgene may be administered to a patient, preferably suspended in abiologically compatible solution or pharmaceutically acceptable deliveryvehicle. A suitable vehicle includes sterile saline. Other aqueous andnon-aqueous isotonic sterile injection solutions and aqueous andnon-aqueous sterile suspensions known to be pharmaceutically acceptablecarriers and well known to those of skill in the art may be employed forthis purpose.

The viral vectors are administered in sufficient amounts to transfectthe cells and to provide sufficient levels of gene transfer andexpression to provide a therapeutic benefit without undue adverseeffects, or with medically acceptable physiological effects, which canbe determined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the liver, oral, intranasal,intravenous, intramuscular, subcutaneous, intradermal, and otherparental routes of administration. Routes of administration may becombined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about1 ml to about 100 ml of solution containing concentrations of from about1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage may beabout 1×10¹³ to 1×10¹⁶ AAV genomes. The dosage will be adjusted tobalance the therapeutic benefit against any side effects and suchdosages may vary depending upon the therapeutic application for whichthe recombinant vector is employed. The levels of expression of thetransgene can be monitored to determine the frequency of dosageresulting in viral vectors, preferably AAV vectors containing theminigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention. For in vitro production, a desiredprotein may be obtained from a desired culture following transfection ofhost cells with a rAAV containing the gene encoding the desired proteinand culturing the cell culture under conditions which permitsexpression. The expressed protein may then be purified and isolated, asdesired. Suitable techniques for transfection, cell culturing,purification, and isolation are known to those of skill in the art.

The following examples illustrate several aspects and embodiments of theinvention.

Example 1 Generation of Infectious Clone of AAV-1

The replicated form DNA of AAV-1 was extracted from 293 cells that wereinfected by AAV-1 and wild type adenovirus type 5.

A. Cell Culture and Virus

AAV-free 293 cells and 84-31 cells were provided by the humanapplication laboratory of the University of Pennsylvania. These cellswere cultured in Dulbecco's Modified Eagle Medium with 10% fetal bovineserum (Hyclone), penicillin (100 U/ml) and streptomycin at 37° C. in amoisturized environment supplied with 5% CO₂. The 84-31 cell lineconstitutively expresses adenovirus genes E1a, E1b, E4/ORF6, and hasbeen described previously [K. J. Fisher, J. Virol., 70:520-532 (1996)].AAV-1 (ATCC VR-645) seed stock was purchased from American Type CultureCollection (ATCC, Manassas, Va.). AAV viruses were propagated in 293cells with wild type Ad5 as a helper virus.

B. Recombinant AAV Generation

The recombinant AAV viruses were generated by transfection using anadenovirus free method. Briefly, the cis plasmid (with AAV ITR), transplasmid (with AAV rep gene and cap gene) and helper plasmid (pFΔ13, withessential regions from the adenovirus genome) were simultaneouslyco-transfected into 293 cells in a ratio of 1:1:2 by calcium phosphateprecipitation. The pFΔ13 helper plasmid has an 8 kb deletion in theadenovirus E2B region and has deletions in most of the late genes. Thishelper plasmid was generated by deleting the RsrII fragment from pFG140(Microbix, Canada). Typically, 50 μg of DNA (cis:trans:PFA13 at ratiosof 1:1:2, respectively) was transfected onto a 15 cm tissue culturedish. The cells were harvested 96 hours post-transfection, sonicated andtreated with 0.5% sodium deoxycholate (37° C. for 10 min). Cell lysateswere then subjected to two rounds of a CsCl gradient. Peak fractionscontaining AAV vector were collected, pooled, and dialyzed against PBSbefore injecting into animals. To make rAAV virus with AAV-1 virion, thepAV1H or p5E18 (2/1) was used as the trans plasmid to provide rep andcap function.

For the generation of rAAV based on AAV-2, p5E18 was used as the transplasmid since it greatly improved the rAAV yield. This plasmid,p5E18(2/2), expresses AAV-2 Rep and Cap and contains a P5 promoterrelocated to a position 3′ to the Cap gene, thereby minimizingexpression of Rep78 and Rep68. The strategy was initially described byLi et al, J. Virol., 71:5236-5243 (1997). P5E18(2/2) was constructed inthe following way. The previously described pMMTV-trans vector (i.e.,the mouse mammary tumor virus promoter substituted for the P5 promoterin an AAV-2-based vector) was digested with Smal and Clal, filled inwith the Klenow enzyme, and then recircularized with DNA ligase. Theresulting construct was digested with XbaI, filled in, and ligated tothe blunt-ended BamHI-XbaI fragment from pCR-p5, constructed in thefollowing way. The P5 promoter of AAV was amplified by PCR and theamplified fragment was subsequently cloned into pCR2.1 (Invitrogen) toyield pCR-P5. The helper plasmid pAV1H was constructed by cloning theBfaI fragment of pAAV-2 into pBluescript II-SK(+) at the BcorV and Smalsites. The 3.0-kb XbaI-KpnI fragment from p5E18(2/2), the 2.3-kbXbaI-KpnI fragment from pAV1H, and the 1.7-kb KpnI fragment fromp5E18(2/2) were incorporated into a separate plasmid P5E18(2/1), whichcontains AAV-2 Rep, AAV-1 Cap, and the AAV-2 P5 promoter located 3′ tothe Cap gene. Plasmid p5E18(2/1) produced 10- to 20-fold higherquantities of the vector than pAV1H (i.e., 10¹² genomes/50 15-cm²plates).

C. DNA Techniques

Hirt DNA extraction was performed as described in the art with minormodification [R. J. Samulski et al., Cell, 33:135-143 (1983)]. Moreparticularly, Hirst solution without SDS was used instead of usingoriginal Hirt solution containing SDS. The amount of SDS present in theoriginal Hirst solution was added after the cells had been fullysuspended. To construct AAV-1 infectious clone, the Hirt DNA from AAV-1infected 293 cells was repaired with Klenow enzyme (New England Biolabs)to ensure the ends were blunt. The treated AAV-1 Hirt DNA was thendigested with BamHI and cloned into three vectors, respectively. Theinternal BamHI was cloned into pBlueScript II-SK+ cut with BamHI to getpAV1-BM. The left and right fragments were cloned into pBlueScriptII-SK+ cut with BamHI+EcoRV to obtain pAV1-BL and pAV1-BR, respectively.The AAV sequence in these three plasmids were subsequently assembledinto the same vector to get AAV-1 infectious clone pAAV-1. The helperplasmid for recombinant AAV-1 virus generation was constructed bycloning the Bfa I fragment of pAAV-1 into pBlueScript II-SK+ at theEcoRV site.

Analysis of the Hirt DNA revealed three bands, a dimer at 9.4 kb, amonomer at 4.7 kb and single-stranded DNA at 1.7 kb, which correlated todifferent replication forms of AAV-1. The monomer band was excised fromthe gel and then digested with BamHI. This resulted in three fragmentsof 1.1 kb, 0.8 kb and 2.8 kb. This pattern is in accordance with thedescription by Bantel-schaal and zur Hausen, Virol., 134(1):52-63(1984). The 1.1 kb and 2.8 kb BamHI fragments were cloned intopBlueScript-KS(+) at BamHI and EcoRV site. The internal 0.8 kb fragmentwas cloned into BamHI site of pBlueScript-KS(+).

These three fragments were then subcloned into the same construct toobtain a plasmid (pAAV-1) that contained the full sequence of AAV-1. ThepAAV-1 was then tested for its ability to rescue from the plasmidbackbone and package infectious virus. The pAAV-1 was then transfectedto 293 cells and supplied with adenovirus type as helper at MOI 10. Thevirus supernatant was used to reinfect 293 cells.

For Southern blot analysis, Hirt DNA was digested with DpnI to removebacteria-borne plasmid and probed with internal BamHI fragment of AAV-1.The membrane was then washed at high stringency conditions, whichincluded: twice 30 minutes with 2×SSC, 0.1% SDS at 65° C. and twice 30minutes with 0.1×SSC, 0.1% SDS at 65° C. The membrane was then analyzedby both phosphor image and X-ray autoradiography. The results confirmedthat pAAV-1 is indeed an infectious clone of AAV serotype 1.

Example 2 Sequencing Analysis of AAV-1

The entire AAV-1 genome was then determined by automatic sequencing andwas found to be 4718 nucleotides in length (FIGS. 1A-1F). Forsequencing, an ABI 373 automatic sequencer as used to determine thesequences for all plasmids and PCR fragments related to this study usingthe FS dye chemistry. All sequences were confirmed by sequencing bothplus and minus strands. These sequences were also confirmed bysequencing two independent clones of pAV-BM, pAV-BL and pAV-BR. Sincethe replicated form of AAV-1 DNA served as the template for sequencedetermination, these sequences were also confirmed by sequencing aseries of PCR products using original AAV-1 seed stock as a template.

The length of AAV-1 was found to be within the range of the otherserotypes: AAV-3 (4726 nucleotides), AAV-4 (4774 nucleotides), AAV-2(4681 nucleotides), and AAV-6 (4683 nucleotides).

The AAV-1 genome exhibited similarities to other serotypes ofadeno-associated viruses. Overall, it shares more than 80% identity withother known AAV viruses as determined by the computer program Megalignusing default settings [DNASTAR, Madison, Wis.]. The key features inAAV-2 can also be found in AAV-1. First, AAV-1 has the same type ofinverted terminal repeat which is capable of forming T-shaped hairpinstructures, despite the differences at the nucleotide level (FIGS. 2 and3). The sequences of right ITRs and left ITRs of AAV-1 are identical.The AAV TR sequence is subdivided into A, A′, B, B′, C, C′, D and D′[Bern, cited above].

These AAV ITR sequences are also virtually the same as those found inAAV-6 right ITR, there being one nucleotide difference in each of A andA′ sequence, and the last nucleotide of the D sequence. Second, theAAV-2 rep binding motif [GCTCGCTCGCTCGCTG (SEQ ID NO: 20)] is wellconserved. Such motif can also be found in the human chromosome 19 AAV-2pre-integration region. Finally, non-structural and structural codingregions, and regulatory elements similar to those of other AAV serotypesalso exist in AAV-1 genome.

Although the overall features of AAV terminal repeats are very muchconserved, the total length of the AAV terminal repeat exhibitsdivergence. The terminal repeat of AAV-1 consists of 143 nucleotideswhile those of AAV-2, AAV-3, and AAV-4 are about 145 or 146 nucleotides.The loop region of AAV-1 ITR most closely resembles that of AAV-4 inthat it also uses TCT instead of the TTT found in AAV-2 and AAV-3. Thepossibility of sequencing error was eliminated using restriction enzymedigestion, since these three nucleotides are part of the SacI site(gagctc; nt 69-74 of SEQ ID NO: 1). The p5 promoter region of AAV-1shows more variations in nucleotide sequences with other AAV serotypes.However, it still maintains the critical regulatory elements. The twocopies of YY1 [See, FIGS. 1A-1F] sites seemed to be preserved in allknown AAV serotypes, which have been shown to be involved in regulatingAAV gene expression. In AAV-4, there are 56 additional nucleotidesinserted between YY1 and E-box/USF site, while in AAV-1, there are 26additional nucleotides inserted before the E-box/USF site. The p19promoter, p40 promoter and polyA can also be identified from the AAV-1genome by analogy to known AAV serotypes, which are also highlyconserved.

Thus, the analysis of AAV terminal repeats of various serotypes showedthat the A and A′ sequence is very much conserved. One of the reasonsmay be the Rep binding motif (GCTC)₃GCTG [SEQ ID NO: 20]. Thesesequences appear to be essential for AAV DNA replication andsite-specific integration. The same sequence has also been shown to bepreserved in a monkey genome [Samulski, personal communication]. Thefirst 8 nucleotides of the D sequence are also identical in all knownAAV serotypes. This is in accordance with the observation of theSrivastava group that only the first 10 nucleotides are essential forAAV packaging [X. S. Wang et al, J. Virol., 71:3077-3082 (1997); X. S.Wang et al, J. Virol., 71:1140-1146 (1997)]. The function of the rest ofthe D sequences still remain unclear. They may be somehow related totheir tissue specificities. The variation of nucleotide in B and Csequence may also suggest that the secondary structure of the ITRs ismore critical for its biological function, which has been demonstratedin many previous publications.

Example 3 Comparison of AAV-1 Sequences

The nucleotide sequences of AAV-1, obtained as described above, werecompared with known AAV sequences, including AAV-2, AAV-4 and AAV-6using DNA Star Megalign. This comparison revealed a stretch of 71identical nucleotides shared by AAV-1, AAV-2 and AAV-6. See, FIGS.1A-1F.

This comparison further suggested that AAV-6 is a hybrid formed byhomologous recombination of AAV-1 and AAV-2. See, FIGS. 3A and 3B. Thesenucleotides divide the AAV-6 genome into two regions. The 5′ half ofAAV-6 of 522 nucleotides is identical to that of AAV-2 except in 2positions. The 3′ half of AAV-6 including the majority of the rep gene,complete cap gene and 3′ ITR is 98% identical to AAV-1.

Biologically, such recombination may enable AAV-1 to acquire the abilityto transmit through the human population. It is also interesting to notethat the ITRs of AAV-6 comprise one AAV-1 ITR and one AAV-2 ITR. Thereplication model of defective parvovirus can maintain this specialarrangement. Studies on AAV integration have shown that a majority ofAAV integrants carries deletions in at least one of the terminalrepeats. These deletions have been shown to be able to be repairedthrough gene conversion using the other intact terminal repeat as atemplate. Therefore, it would be very difficult to maintain AAV-6 as ahomogenous population when an integrated copy of AAV-6 is rescued fromhost cells with helper virus infection. The AAV-6 with two identicalAAV-2 ITRs or two identical AAV-1 ITRs should be the dominant variants.The AAV-6 with two AAV-1 ITRs has been observed by Russell's group[Rutledge, cited above (1998)]. So far there is no report on AAV-6 withtwo AAV-2 ITRs. Acquirement of AAV-2 P5 promoter by AAV-6 may haveexplained that AAV-6 have been isolated from human origin while AAV-1with the same virion has not. The regulation of P5 promoter betweendifferent species of AAV may be different in vivo. This observationsuggests the capsid proteins of AAV were not the only determinants fortissue specificity.

Although it is clear that AAV-6 is a hybrid of AAV-1 and AAV-2, AAV-6has already exhibited divergence from either AAV-1 or AAV-2. There aretwo nucleotide differences between AAV-6 and AAV-2 in their first 450nucleotides. There are about 1% differences between AAV-6 and AAV-1 innucleotide levels from nucleotides 522 to the 3′ end. There also existsa quite divergent region (nucleotide 4486-4593) between AAV-6 and AAV-1(FIGS. 1A-1F). This region does not encode any known proteins for AAVs.These differences in nucleotide sequences may suggest that AAV-6 andAAV-1 have gone through some evolution since the recombination tookplace. Another possible explanation is that there exists another variantof AAV-1 which has yet to be identified. So far, there is no evidence torule out either possibility. It is still unknown if other hybrids (AAV-2to AAV-4, etc.) existed in nature.

The coding region of AAV-1 was deduced by comparison with other knownAAV serotypes. Table 1 illustrates the coding region differences betweenAAV-1 and AAV-6. The amino acid residues are deduced according to AAV-2.

With reference to the amino acid position of AAV-1, Table 1 lists theamino acids of AAV-1 which have been changed to the corresponding onesof AAV-6. The amino acids of AAV-1 are shown to the left of the arrow.Reference may be made to SEQ ID NO: 5 of the amino acid sequence ofAAV-1 Rep 78 and to SEQ ID NO: 13 for the amino acid sequence of AAV-1VP1.

TABLE 1 Coding region variations between AAV-1 and AAV-6 Rep protein(Rep78) Cap protein (VP1) Position(s) Amino acids Position(s) Aminoacids 28 S→N 129 L→F 191 Q→H 418 E→D 192 H→D 531 E→K 308 E→D 584 F→L 598A→V 642 N→H

It was surprising to see that the sequence of the AAV-1 coding region isalmost identical to that of AAV-6 from position 452 to the end of codingregion (99%). The first 508 nucleotides of AAV-6 have been shown to beidentical to those of AAV-2 [Rutledge, cited above (1998)]. Since thecomponents of AAV-6 genome seemed to be AAV-2 left ITR—AAV-2 p5promoter—AAV-1 coding region—AAV-1 right ITR, it was concluded thatAAV-6 is a naturally occurred hybrid between AAV-1 and AAV-2.

Example 4 Gene Therapy Vector Based on AAV-1

Recombinant gene transfer vectors based on AAV-1 viruses wereconstructed by the methods described in Example 1. To produce a hybridrecombinant virus with AAV-1 virion and AAV-2 ITR, the AAV-1 transplasmid (pAV1H) and the AAV-2 cis-lacZ plasmid (with AAV-2 ITR) wereused. The AAV-2 ITR was used in this vector in view of its known abilityto direct site-specific integration. Also constructed for use in thisexperiment was an AAV-1 vector carrying the green fluorescent protein(GFP) marker gene under the control of the immediate early promoter ofCMV using pAV1H as the trans plasmid.

A. rAAV-1 Viruses Transfect Host Cells In Vitro

84-31 cells, which are subclones of 293 cells (which express adenovirusE1a, E1b) which stably express E4/ORFS, were infected with rAAV-1 GFP orrAAV-lacZ. High levels of expression of GFP and lacZ was detected in thecultured 84-31 cells. This suggested that rAAV-1 based vector was verysimilar to AAV-2 based vectors in ability to infect and expressionlevels.

B. rAAV-1 Viruses Transfect Cells In Vivo

The performance of AAV-1 based vectors was also tested in vivo. TherAAV-1 CMV-α1AT virus was constructed as follows. The EcoRI fragment ofpAT85 (ATCC) containing human α1-antitrypsin (α1AT) cDNA fragment wasblunted and cloned into PCR (Promega) at a SmaI site to obtain PCR-α1AT.The CMV promoter was cloned into PCR-α1AT at the XbaI site. The Alb-α1ATexpression cassette was removed by XhoI and Clal and cloned into pAV1Hat the XbaI site. This vector plasmid was used to generateAAV-1-CMV-α1AT virus used in the experiment described below.

For screening human antibodies against AAV, purified AAV virus is lysedwith Ripa buffer (10 mM Tris pH 8.2, 1% Triton X-100, 1% SDS, 0.15 MNaCl) and separated in 10% SDS-PAGE gel. The heat inactivated humanserum was used at a 1 to 1000 dilution in this assay. The rAAV-1CMV-α1AT viruses were injected into Rag-1 mice through tail veininjection at different dosages. The concentration of humanα1-antitrypsin in mouse serum was measured using ELISA. The coatingantibody is rabbit anti-human human α1-antitrypsin (Sigma). Thegoat-antihuman α1-antitrypsin (Sigma) was used as the primary detectionantibodies. The sensitivity of this assay is around 0.3 ng/ml to 30ng/ml. The expression of human α1-antitrypsin in mouse blood can bedetected in a very encouraging level. This result is shown in Table 2.

TABLE 2 Human α1-Antitrypsin Expressed in Mouse Liver Amount of virusinjected Week 2 (ng/ml) Week 4 (ng/ml) 2 × 10¹⁰ genomes 214.2 171.4 1 ×10¹⁰ genomes 117.8 109.8 5 × 10¹⁰ genomes 64.5 67.8 2.5 × 10¹⁰ genomes  30.9 58.4

rAAV-1 CMV-lacZ viruses were also injected into the muscle of C57BL6mice and similar results were obtained. Collectively, these resultssuggested that AAV-1 based vector would be appropriate for both liverand muscle gene delivery.

Example 5 Neutralizing Antibodies Against AAV-1

Simple and quantitative assays for neutralizing antibodies (NAB) toAAV-1 and AAV-2 were developed with recombinant vectors. A total of 33rhesus monkeys and 77 normal human subjects were screened.

1. Nonhuman Primates

Wild-caught juvenile rhesus monkeys were purchased from Covance (Alice,Tex.) and LABS of Virginia (Yemassee, S.C.) and kept in full quarantine.The monkeys weighed approximately 3 to 4 kg. The nonhuman primates usedin the Institute for Human Gene Therapy research program arepurposefully bred in the United States from specific-pathogen-freeclosed colonies. All vendors are US Department of Agriculture class Adealers. The rhesus macaques are therefore not infected with importantsimian pathogens, including the tuberculosis agent, major simianlentiviruses (simian immunodeficiency virus and simian retroviruses),and cercopithecine herpesvirus. The animals are also free of internaland external parasites. The excellent health status of these premiumanimals minimized the potential for extraneous variables. For thisstudy, serum was obtained from monkeys prior to initiation of anyprotocol.

NAB titers were analyzed by assessing the ability of serum antibody toinhibit the transduction of reporter virus expressing green fluorescentprotein (GFP) (AAV1-GFP or AAV2-GFP) into 84-31 cells. Various dilutionsof antibodies preincubated with reporter virus for 1 hour at 37° C. wereadded to 90% confluent cell cultures. Cells were incubated for 48 hoursand the expression of green fluorescent protein was measured byFluorolmaging (Molecular Dynamics). NAB titers were calculated as thehighest dilution at which 50% of the cells stained green.

Analysis of NAB in rhesus monkeys showed that 61% of animals testedpositive for AAV-1; a minority (24%) has NAB to AAV-2. Over one-third ofanimals had antibodies to AAV-1 but not AAV-2 (i.e., were monospecificfor AAV-1), whereas no animals were positive for AAV-2 without reactingto AAV-1. These data support the hypothesis that AAV-1 is endemic inrhesus monkeys. The presence of true AAV-2 infections in this group ofnonhuman primates is less clear, since cross-neutralizing activity of anAAV-1 response to AAV-2 can not be ruled out. It is interesting thatthere is a linear relationship between AAV-2 NAB and AAV-1 NAB inanimals that had both.

2. Humans

For these neutralization antibody assays, human serum samples wereincubated at 56° C. for 30 min to inactivate complement and then dilutedin DMEM. The virus (rAAV or rAd with either lacZ or GFP) was then mixedwith each serum dilution (20×, 400×, 2000×, 4000×, etc.) and incubatedfor 1 hour at 37° C. before applied to 90% confluent cultures of 84-31cells (for AAV) or Hela cells (for adenovirus) in 96-well plates. After60 minutes of incubation at culture condition, 100 μl additional mediacontaining 20% FCS was added to make final culture media containing 10%FCS.

The results are summarized in Table 3.

TABLE 3 Adenovirus AAV-1 AAV-2 # of samples Percentage − − − 41 53.2%  +− − 16 20.8%  − + − 0 0.0% − − + 2 2.6% − + + 2 2.6% + − + 3 3.9% + + −0 0.0% + + + 13 16.9%  Total 77 100% 

The human neutralizing antibodies against these three viruses seemed tobe unrelated since the existence of neutralizing antibodies against AAVare not indications for antibodies against adenovirus. However, AAVrequires adenovirus as helper virus, in most of the cases, theneutralizing antibodies against AAV correlated with the existence ofneutralizing antibodies to adenovirus. Among the 77 human serum samplesscreened, 41% of the samples can neutralize the infectivity ofrecombinant adenovirus based on Ad5. 15/77 (19%) of serum samples canneutralize the transduction of rAAV-1 while 20/77 (20%) of the samplesinhibit rAAV-2 transduction at 1 to 80 dilutions or higher. All serumsamples positive in neutralizing antibodies for AAV-1 in are alsopositive for AAV-2. However, there are five (6%) rAAV-2 positive samplesthat failed to neutralize rAAV-1. In samples that are positive forneutralizing antibodies, the titer of antibodies also varied in thepositive ones. The results from screening human sera for antibodiesagainst AAVs supported the conclusion that AAV-1 presents the sameepitome as that of AAV-2 to interact with cellular receptors since AAV-1neutralizing human serums can also decrease the infectivity of AAV-2.However, the profile of neutralizing antibodies for these AAVs is notidentical, there are additional specific receptors for each AAVserotype.

Example 6 Recombinant AAV Viruses Exhibit Tissue Tropism

The recombinant AAV-1 vectors of the invention and the recombinant AAV-2vectors [containing the gene encoding human α1-antitrypsin (α1AT) ormurine erythropoietin (Epo) from a cytomegalovirus-enhanced β-actinpromoter (CB)] were evaluated in a direct comparison to equivalentcopies of AAV-2 vectors containing the same vector genes.

Recombinant viruses with AAV-1 capsids were constructed using thetechniques in Example 1. To make rAAV with AAV-1 virions, pAV1H or p5E18(2/1) was used as the trans plasmid to provide Rep and Cap functions.For the generation of the rAAV based on AAV-2, p5E18(2/2) was used asthe trans plasmid, since it greatly improved the rAAV yield. [Earlyexperiments indicated similar in vivo performances of AAV-1 vectorsproduced with pAV1H and p5E19 (2/1). All subsequent studies used AAV-1vectors derived from p5E18(2/1) because of the increased yield.]

Equivalent stocks of the AAV-1 and AAV-2 vectors were injectedintramuscularly (5×10¹⁰ genomes) or liver via the portal circulation(1×10¹¹ genomes) into immunodeficient mice, and the animals (fourgroups) were analyzed on day 30 for expression of transgene. See, FIGS.4A and 4B.

AAV-2 vectors consistently produced 10- to 50-fold more serumerythropoietin or α1-antitrypsin when injected into liver compared tomuscle. (However, the AAV-1-delivered genes did achieve acceptableexpression levels in the liver.) This result was very different fromthat for AAV-1 vectors, with which muscle expression was equivalent toor greater than liver expression. In fact, AAV-1 outperformed AAV-2 inmuscle when equivalent titers based on genomes were administered.

Example 7 Gene Delivery Via rAAV-1

C57BL/6 mice (6- to 8-week old males, Jackson Laboratories) wereanalyzed for AAV mediated gene transfer to liver following intrasplenicinjection of vector (i.e., targeted to liver). A total of 10¹¹ genomeequivalents of rAAV-1 or rAAV-2 vector were injected into thecirculation in 100 μl buffered saline. The first vector contained eitheran AAV-1 capsid or an AAV-2 capsid and expressed α1AT under the controlof the chicken β-actin (CB) promoter. Day 28 sera were analyzed forantibodies against AAV-1 or AAV-2 and serum α1AT levels were checked.Animals were then injected with an AAV-1 or AAV-2 construct expressingerythropoietin (Epo, also under the control of the CB promoter). Onemonth later sera was analyzed for serum levels of Epo. The followinggroups were analyzed (FIGS. 5A-5D).

In Group 1, vector 1 was AAV-2 expressing α1AT and vector 2 was AAV-2expressing Epo. Animals generated antibodies against AAV-2 following thefirst vector administration which prevented the readministration of theAAV-2 based vector. There was no evidence for cross-neutralizing theantibody to AAV-1.

In Group 2, vector 1 was AAV-1 expressing α1AT while vector 2 was AAV-1expressing Epo. The first vector administration did result insignificant α1AT expression at one month associated with antibodies toneutralizing antibodies to AAV-1. The animals were not successfullyreadministered with the AAV-1 Epo expressing construct.

In Group 3, the effectiveness of an AAV-2 vector expressing Epo injectedinto a naive animal was measured. The animals were injected with PBS andinjected with AAV-2 Epo vector at day 28 and analyzed for Epo expressionone month later. The neutralizing antibodies were evaluated at day 28 sowe did not expect to see anything since they received PBS with the firstvector injection. This shows that in naive animals AAV-2 is veryefficient at transferring the Epo gene as demonstrated by high level ofserum Epo one month later.

Group 4 was an experiment similar to Group 3 in which the animalsoriginally received PBS for vector 1 and then the AAV-1 expressing Epoconstruct 28 days later. At the time of vector injection, thereobviously were no antibodies to either AAV-1 or AAV-2. The AAV-1 basedvector was capable of generating significant expression of Epo whenmeasured one month later.

Group 5 is a cross-over experiment where the initial vector is AAV-2expressing α1AT followed by the AAV-1 construct expressing Epo. Theanimals, as expected, were efficiently infected with the AAV-2 vectorexpressing α1AT as shown by high levels of the protein in blood at 28days. This was associated with significant neutralizing antibodies toAAV-2. Importantly, the animals were successfully administered AAV-1following the AAV-2 vector as shown by the presence of Epo in serum 28days following the second vector administration. At the time of thisvector administration, there was high level AAV-2 neutralizingantibodies and very low cross-reaction to AAV-1. The level of Epo wasslightly diminished possibly due to a small amount of cross-reactivity.Group 6 was the opposite cross-over experiment in which the initialvector was AAV-1 based, whereas the second experiment was AAV-2 based.The AAV-1 vector did lead to significant gene expression of α1AT, whichalso resulted in high level AAV-1 neutralizing antibody. The animalswere very efficiently administered AAV-2 following the initial AAV-1vector as evidenced by high level Epo.

A substantially identical experiment was performed in muscle in which5×10¹⁰ genomes were injected into the tibialis anterior of C57BL/6 miceas a model for muscle directed gene therapy. The results are illustratedin FIGS. 6A-6D and are essentially the same as for liver.

In summary, this experiment demonstrates the utility of using an AAV-1vector in patients who have pre-existing antibodies to AAV-2 or who hadinitially received an AAV-2 vector and need readministration.

Example 8 Construction of Recombinant Viruses Containing AAV-1 ITRs

This example illustrates the construction of recombinant AAV vectorswhich contain AAV-1 ITRs of the invention.

An AAV-1 cis plasmid is constructed as follows. A 160 bp Xho-NruI AAV-1fragment containing the AAV-1 5′ ITR is obtained from pAV1-BL. pAV1-BLwas generated as described in Example 1. The Xho-NruI fragment is thencloned into a second pAV1-BL plasmid at an XbaI site to provide theplasmid with two AAV-1 ITRs. The desired transgene is then cloned intothe modified pAV-1BL at the NruI and BamHI site, which is locatedbetween the AAV-1 ITR sequences. The resulting AAV-1 cis plasmidcontains AAV-1 ITRs flanking the transgene and lacks functional AAV-1rep and cap.

Recombinant AAV is produced by simultaneously transfecting threeplasmids into 293 cells. These include the AAV-1 cis plasmid describedabove; a trans plasmid which provides AAV rep/cap functions and lacksAAV ITRs; and a plasmid providing adenovirus helper functions. The repand/or cap functions may be provided in trans by AAV-1 or another AAVserotype, depending on the immunity profile of the intended recipient.Alternatively, the rep or cap functions may be provided in cis by AAV-1or another serotype, again depending on the patient's immunity profile.

In a typical cotransfection, 50 μg of DNA (cis:trans:helper at ratios of1:1:2, respectively) is transfected onto a 15 cm tissue culture dish.Cells are harvested 96 hours post transfection, sonicated and treatedwith 0.5% sodium deoxycholate (37° for 10 min) Cell lysates are thensubjected to 2-3 rounds of ultracentrifugation in a cesium gradient.Peak fractions containing rAAV are collected, pooled and dialyzedagainst PBS. A typical yield is 1×10¹³ genomes/10⁹ cells.

Using this method, one recombinant virus construct is prepared whichcontains the AAV-1 ITRs flanking the transgene, with an AAV-1 capsid.Another recombinant virus construct is prepared with contains the AAV-1ITRs flanking the transgene, with an AAV-2 capsid.

All publications cited in this specification are incorporated herein byreference. While the invention has been described with reference to aparticularly preferred embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe claims.

What is claimed is:
 1. A recombinant virus having an AAV-1 capsidcomprising a vp1 protein, a vp2 protein, and a vp3 protein, wherein saidvp3 protein has an amino acid sequence of SEQ ID NO:17, wherein saidrecombinant virus further comprises a heterologous molecule whichcomprises an AAV 5′ inverted terminal repeat sequence (ITR), atransgene, and an AAV 3′ ITR.
 2. The recombinant virus according toclaim 1, wherein said vp1 protein has the amino acid sequence of SEQ IDNO:
 13. 3. The recombinant virus according to claim 1, wherein said vp2protein has the amino acid sequence of SEQ ID NO:
 15. 4. The recombinantvirus according to claim 1, wherein the 5′ ITR and 3′ ITR are of AAVserotype
 2. 5. The recombinant virus according to claim 1, wherein the5′ ITR and 3′ ITR are of AAV serotype
 1. 6. The recombinant virusaccording to claim 1, further comprising a promoter which directsexpression of the transgene.
 7. The recombinant virus according to claim1, wherein said transgene encodes a protein or peptide.
 8. Therecombinant virus according to claim 7, wherein said protein or peptideis a therapeutic protein or peptide.
 9. The recombinant virus accordingto claim 7, wherein said protein or peptide is an immunogenic protein orpeptide.
 10. The recombinant virus according to claim 1, wherein saidtransgene encodes a cytokine, a hormone, or a growth factor.
 11. Therecombinant virus according to claim 1, wherein said transgene is alpha1 anti-trypsin (α1AT).
 12. The recombinant virus according to claim 1,wherein said transgene is erythropoietin (epo).
 13. An isolated hostcell transformed with the recombinant virus of claim
 1. 14. Acomposition comprising a recombinant virus according to claim 1 and apharmaceutically acceptable carrier.
 15. The composition according toclaim 14, wherein said composition is formulated for delivery to muscle.16. The composition according to claim 14, wherein said composition isformulated for delivery to liver.
 17. The composition according to claim14, wherein said composition is formulated for intranasal delivery.